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Am J Physiol Endocrinol Metab 294: E640-E642, 2008. doi:10.1152/ajpendo.00797.2007
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LETTERS TO THE EDITOR

The interpretation of abnormal 31P magnetic resonance saturation transfer measurements of Pi/ATP exchange in insulin-resistant skeletal muscle

Graham J. Kemp

School of Clinical Science, Faculty of Medicine, University of Liverpool, Liverpool, United Kingdom

TO THE EDITOR: To study relationships between muscle mitochondrial function, fatty acid oxidation, intramyocellular lipid (IMCL), and insulin resistance (IR), Laurent et al. (21a) measured Pi/ATP exchange by 31P magnetic resonance saturation (MRS) transfer (ST) in resting rat muscle and found that it decreased transiently by high dietary fat (HF) intake. Following precedent, they identified Pi/ATP exchange with mitochondrial ATP synthesis, noting that "actual rates obtained ... in rats at rest are in excellent agreement with recent human data," and took the decrease as evidence of mitochondrial dysfunction (21a). I would like to make two points about absolute rates and pathophysiological logic.

Absolute Rates

Given the allometry of basal metabolic rate (34), to which it makes a large contribution (33), resting muscle ATP turnover should presumably scale with weight(–0.25), making it approximately four times greater in rat (~7 in mouse) than in human. Unlike Pi/ATP exchange (21a), published measurements by several other methods seem roughly consistent with this (Fig. 1A). Moreover, although "there is no gold standard against which the ATP synthesis rate measured by 31P saturation transfer can easily be validated" (21a), the estimates it gives are notably high (Fig. 1A). For Pi/ATP exchange to validly measure mitochondrial ATP synthesis (i.e., net flux through F1F0-ATPase) requires this to be far from equilibrium [as measurements in vitro support, at least at high turnover (21)] and glycolytic Pi/ATP exchange (catalyzed by glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase) to be small (7, 15, 21a, 32). No direct comparisons have been made, but suprabasal Pi/ATP exchange rates in stimulated rat muscle were noted (7) to be approximately consistent with O2 consumption (12) and phosphocreatine (PCr) kinetic (35) data and with poststimulation PCr resynthesis rates (38). However, upon reexamination (Fig. 1B) these comparisons do not rule out appreciable glycolytic Pi/ATP exchange. If skeletal muscle resembles heart, where this discrepancy is broadly independent of ATP turnover (32) (in effect, a constant error), the 20–40% Pi/ATP exchange defect in HF (21a) and other IR states (4, 28, 29, 36) presumably underestimates that in ATP synthesis. However, the similar percent defects in Pi/ATP exchange and tricarboxylic cycle (TCAC) flux measured by 13C MRS in IR (4, 28, 29) suggest that the discrepancy is turnover dependent (a proportional error). This is not resolved by the observations that triiodothyronine (14, 22) and 2,4-dinitrophenol (14) decrease, whereas uncoupling protein-3 knockout increases (8), the ratio of Pi/ATP exchange to TCAC flux [a qualitative index of coupling (15, 16)] or that 2,4-dinitrophenol does not affect directly measured glycolytic exchange (14).


Figure 1
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  		Fig. 1. Estimates of muscle oxidative ATP turnover (mmol·l cell water–1·min–1) by various methods. A: resting rates in healthy human, rat, and mouse muscle. Each point is 1 published mean: 1 = 31P magnetic resonance saturation (MRS) transfer (ST) Pi/ATP exchange in human (6, 22, 28, 29, 36), rat (7, 14–16, 21a), and mouse (8) [Laurent et al. (21a) reported the highest rat value]; 2 = 13C MRS tricarboxylic cycle (TCAC) rate in human (4, 22, 28), rat (13–16), and mouse (8); 3 = arteriovenous-difference (AVD) VO2 in human (3, 10, 39) and rat (2, 12, 33); 4 = near-infrared spectroscopy (NIRS) [O2] kinetics during acute ischemia in human (1, 39) and mouse (23–25); 5 = 31P MRS phosphocreatine (PCr) kinetics during acute ischemia in human (1, 5, 39) and mouse (24, 25); 6 = [15O]O2 positron emission tomography (PET) in human (citations are exhaustive for 31P MRS ST, 13C MRS, and mouse studies; otherwise they are representative; Ref. 39 is cited as a compendium) (11, 17, 27). Assumptions: P:O = 2.16 from mouse ischemic 31P MRS/NIRS (shown as linked points) (24, 25); human leg muscle mass = 10 kg; VO2/TCAC flux = 3, and 1 mol O2 = 25.5 l (10); wet/dry = 4.35, and cell water = 0.67 l/kg (7). Dashed lines give overall means of published values, for which rat/human ratio is ~3:5 by VO2 and 13C MRS (cf expected ~4) and mouse/human ratio is ~4:7 by ischemic 31P MRS/NIRS and 13C MRS (cf expected ~7); Pi/ATP exchange is similar in human and rat (21a) but lower in the single mouse study (8). B: rates in rat muscle vs. PCr concentration ([PCr]) relative to resting values. References are indicated in the figure. Comparisons of stimulated muscle ST Pi/ATP exchange (7) with contraction-cost predictions from VO2 (12) and PCr kinetics (35) are modified from Ref. 7. Initial poststimulation PCr resynthesis rate (38) approximates suprabasal oxidative ATP synthesis (5) but is given here as total oxidative ATP synthesis using basal VO2 (12). Note the close match of PCr recovery to interpolated VO2 data (12) and similarity (38) to suprabasal Pi/ATP exchange (7), except for the basal discrepancy.

 
Flux Vs. Capacity, Supply Vs. Demand

The distinction between flux and capacity is why "maximal enzyme activities do not necessarily reflect actual flux through metabolic pathways" (21a, 29). On the conventional [but perhaps simplistic (8)] understanding of muscle ATP synthesis as demand driven, with the creatine kinase equilibrium acting as feedback adjuster of ATP supply (18, 40), the observation of decreased resting ATP turnover in HF (21a) and other IR states (4, 28, 29, 36) is evidence per se of decreased ATP demand, not "defects in mitochondrial oxidative capacity (i.e., due to mitochondrial dysfunction and/or mitochondrial loss)," plausible though this is, given ex vivo evidence of mitochondrial abnormalities (21a, 29). In the case of mitochondrial loss (28), or mitochondrial dysfunction similarly conceptualized (19), normal ATP turnover is maintained by increased error signals such as cytosolic ADP concentration ([ADP]) and Pi concentration ([Pi]) (18, 40), both of which respond to ATP supply/demand mismatch, although steady-state [Pi] change requires altered sarcolemmal Pi transport (31). Are there insulin-related changes in mitochondrial functional capacity as well as in ATP synthesis rate? Decreased Pi/ATP exchange in HF (21a) and other IR states (4, 28, 29, 36) and its insulin stimulation in normal muscle (6, 30) are often due to changes in [Pi] (21a, 29) rather than in the ST rate constant, i.e., (Pi/ATP exchange)/[Pi] (7, 21a). If Pi/ATP exchange does reflect mitochondrial ATP synthesis, a linear relation to [Pi] [at least below its effective Km (40)] could be interpreted, like the linear relation to {Delta}[PCr] of the PCr recovery data in Fig. 1B, as consistent with a fixed mitochondrial capacity (26) conceptualized as the inferred maximal rate of oxidative ATP synthesis at high [ADP] and [Pi] (5, 18, 19). Increase in resting [Pi] (30, 37) by insulin stimulation of sarcolemmal Pi uptake (31) is an external perturbation of a feedback signal that cannot alter steady-state flux, but insulin also stimulates ATP demand (6), and so increased [Pi] could be seen as a feedback mediator adjusting the ATP synthesis rate upward. Conversely, decreased ATP synthesis rate in HF (21a) and other IR states (4, 28, 29, 36) might be matched to decreased ATP demand partly by low [Pi] resulting from reduced sarcolemmal Pi uptake. But this ignores [ADP], calculable from pH and [PCr] but often unreported. Insulin does not change [PCr] (30, 37) but decreases pH (37), which tends to lower [ADP], working against the tendency of a rise in [Pi] to increase the free energy of ATP hydrolysis ({Delta}GATP), a quantity that integrates the feedback effects of [Pi] and [ADP] in the nonequilibrium thermodynamic approach to mitochondrial flux control (18). Insulin-stimulated ATP synthesis (6, 30) at unchanged or decreased {Delta}GATP would imply an increase in functional mitochondrial capacity that is conceptually similar to "parallel activation" (20). Conversely, decreased ATP synthesis in HF (21a) and other IR states (4, 28, 29, 36) at unchanged or increased {Delta}GATP would indeed imply mitochondrial loss or impairment in addition to decreased ATP demand. Resolving this will necessitate more measurements of [Pi] and [ADP] and a better understanding of their mitochondrial effects (18, 40) in resting muscle. [Although less extreme extrapolation is required to estimate mitochondrial capacity from postexercise recovery kinetics (19, 26, 38), the argument is complicated by possible microvascular disease.] The present significance of these distinctions is that, although transient IMCL increase results from temporary excess of fatty acid supply over fat oxidation, at steady state both rates must be equal, and high IMCL can be sustained only by increased fatty acid supply and/or decreased fat oxidative capacity (so to speak, the apparent rate constant of fat oxidation). Laurent et al. (21a) have shown that HF, which increases fatty acid supply and promotes IMCL accumulation, very likely causes a decrease in mitochondrial ATP synthesis rate, presumably secondary to decreased overall ATP demand, and perhaps implies a decrease in fat oxidation rate.

FOOTNOTES


Address for reprint requests and other correspondence: G. J. Kemp, School of Clinical Science, Faculty of Medicine, Univ. of Liverpool, Liverpool L69 36A, UK (e-mail: gkemp{at}liv.ac.uk)

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D. Laurent
Reply to Kemp: a clarification on the interpretation of muscular ATP synthase flux data obtained by 31P saturation transfer
Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E643 - E644.
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